The long term objective of this project is to develop methods to therapeutically target defects in pre-mRNA splicing that cause human disease. One disease that can potentially be cured by targeting pre- mRNA splicing is spinal muscular atrophy (SMA). SMA is a pediatric neurodegenerative disease for which there is currently no cure or effective therapy. The disease is caused by the homozygous loss of the survival of motor neuron, SMN1, gene. Humans have a second gene, SMN2, which is nearly identical to SMN1. Both SMN1 and SMN2 code for SMN protein. However, the majority of SMN2 mRNA transcripts splice out exon 7. This alternative mRNA isoform codes for a truncated, unstable protein. Thus, SMA results from the reduction of SMN protein levels caused by the loss of SMN1. The presence of SMN2 in most SMA patients provides a unique opportunity to treat the disease by increasing SMN2 exon 7 splicing and thereby restoring SMN levels. We have recently identified a tetracycline derivative that improves SMN protein levels by directly targeting the splicing reaction to increase SMN2 exon 7 splicing. This compound is particularly attractive as a therapeutic due to the favorable pharmacokinetic characteristics of the tetracycline family of molecules and because the compound appears to act at a very specific step in the gene expression pathway. The goal of our application is to develop this potent activator of SMN2 splicing as a therapeutic for the treatment of SMA and potentially other diseases caused by splicing defects. The central hypothesis of the study is that targeting defective splicing in diseases such as SMA will lead to a therapeutic increase in protein expression. Aim 1 of this study is to use induced pluripotent stem cells derived from SMA patients to determine the effectiveness of splicing effector molecules, such as the tetracycline derivatives, in rescuing motor neuron degeneration and also to characterize the cellular consequences of SMN protein loss during motor neuron degeneration. Aim 2 is to identify the molecular target and mechanism of action by which tetracycline derivatives increase SMN2 splicing. Aim 3 is to discover novel targets and develop alternative strategies for SMA therapy. The proposed experiments will provide insights and therapeutics for the treatment of SMA and will also advance therapeutic approaches to treat other diseases caused by splicing defects. These studies will also have a broad impact on our understanding of induced pluripotent stem cells as models for human disease and the cellular defects of SMN protein loss in motor neurons of SMA patients. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to discover and develop effective therapeutics for the treatment of the pediatric neurodegenerative disease, spinal muscular atrophy (SMA), and other human diseases that have common molecular defects for which there is currently no cure. Compounds based on the structure of the antibiotic tetracycline have been identified as modulators of a defect in spinal muscular atrophy. This class of drug is relatively safe and would be an ideal therapeutic for the treatment of disease.